I. The Gly269 -> Ser mutation in the beta-hexosaminidase alpha-subunit has been found in homozygous form in 3 non-Jewish patients with adult GM2 gangliosidosis. This is in contrast to the situation in Ashkenazi Jewish patients where the Gly269 -> Ser mutation is present but always in compound heterozygosity with a null Tay-Sachs allele. II. - We have isolated near full length cDNAs encoding alpha and beta-subunits of murine P-bexosaminidase. The nucleotide sequence of the alpha-subunit has been determined. These cDNAs have been used to isolate genomic clones encompassing portions of the murine alpha and beta-subunit genes. III. The human alpha and beta-subunits have been incorporated into baculovirus vectors which have been used to overexpress beta-hexosaminidase B (an alpha-subunit dimer) and P-hexosaminidase S (an alpha-subunit dimer) . The enzymes have been purified from culture medium of recombinant virus-infected SF9 cells by Concanavalin A affinity chromatography followed by Mono S column chromatography, for the B isozyme, or phenyl-Superose column chromatography for the -S isozyme. IV. A rat endoplasmic reticulum carboxyl esterase has been cloned and sequenced. Other partial cDNAs from this gene family have also been sequenced. The full-length cDNA and mutated versions have been expressed in COS 1 cells in order to identify the protein determinant that signifies retention in the endoplasmic reticulum.